The accurate transmission of genetic material during cell division depends on the ability of the cell to coordinate the various processes involved in chromosome segregation. This is accomplished by a network of regulatory mechanisms whose function is to ensure that late cell cycle steps are executed only after earlier ones have been successfully completed. We study the regulation of cell cycle progression through mitosis, using the budding yeast Saccharomyces cerevisiae as a model system. We have previously shown that an anaphase inhibitor, the Pds1 protein, acts as a key regulator of mitotic events; it controls the timing of anaphase initiation and it prevents the premature exit from mitosis. Previous work has demonstrated that Pds1 associates with an anaphase activator, the Esp1 protein. The outcome of this interaction is two fold: Pds1 serves as an inhibitor of Esp1 but it is also required for Esp1?s nuclear localization. The inhibition by Pds1 is alleviated by its degradation in a process that involves the anaphase promoting complex ubiquitin ligase (APC), but the mechanistic details of this process have been obscure. Whether Pds1 has additional targets is currently unknown, but its involvement in regulating mitotic exit suggests that additional targets do exist. Although Pds1 is not required for cell viability under normal growth conditions, it becomes essential once cells are challenged with intracellular damage and must delay the progression through mitosis. To understand how Pds1 carries out its role as a mitotic inhibitor and to identify additional components of this regulatory pathway we have been engaged in the following experimental approaches: (1) A genetic screen aimed at identifying mutants that abrogate the ability of Pds1 to induce a mitotic arrest. This screen is expected to reveal proteins that interact directly with Pds1 as well as downstream components of this regulatory pathway. In the past year we have been focusing on a class of mutants that fail to inhibit the exit of mitosis in the absence of anaphase and go on to divide without chromosome segregation. (2) A yeast two-hybrid screen for identifying Pds1-interacting proteins. In this screen we have so far identified one Pds1-interacting protein, the Cdc20 protein. Cdc20 associates with the APC and provides substrate specificity. Our findings suggest that Pds1 interacts directly with Cdc20 and they led us to the understanding of how Pds1 is being recognized by the APC (published in Hilioti et al, Curr Biol 11, 1347-1352, 2001) (3) The role of Cdc28-mediated phosphorylation of Pds1. Through our biochemical analysis of the Pds1 protein we discovered that Pds1 is phosphorylated by the cyclin-dependent kinase Cdc28. The phosphorylation sites were mapped and mutated. Pds1 forms that cannot undergo Cdc28-mediated phosphorylation cannot interact with Pds1?s target, the Esp1 protein, and as a result Esp1 fails to localize to the nucleus. Our work has uncovered a novel regulatory mechanism that regulates the Pds1-Esp1 interaction. We propose that the release of Esp1 from Pds1?s inhibitory action can take place by one of two mechanisms: Pds1 degradation via the APC pathway, and Pds1?s dephosphorylation by a yet unknown phosphatase. We are now concentrating our efforts in determining the nature of this predicted dephosphorylation activity. (4) The nuclear targeting of Esp1. Pds1 is not an essential protein and thus we assume that Esp1, which is essential, must have a Pds1-independent route into the nucleus. To characterize this pathway we are carrying out a screen for mutants that loose viability in the absence of Pds1. Our working hypothesis is that if we abolish both the Pds1-dependent and independent pathways for Esp1?s nuclear import the cells will not be able to survive. Other mutants that may come out of this screen are those that lead to DNA or spindle damage and thus requires Pds1?s checkpoint function for viability.